There are 15.7 million people in the United States who have diabetes, which is the seventh leading cause of death in this country. As a chronic disease that has no cure, diabetes is one of the most costly health problems in America. Health care and other costs directly related to diabetes treatment, as well as the costs of lost productivity, run $92 billion annually.
Type I autoimmune diabetes results from the destruction of insulin producing beta cells in the pancreatic islets of Langerhans. The adult pancreas has very limited regenerative potential, and so these islets are not replaced after they are destroyed. The patient""s survival then depends on exogenous administration of insulin. There are an estimated 500,000 to 1 million people with type 1 diabetes in the United States today. The risk of developing type 1 diabetes is higher than virtually all other severe chronic diseases of childhood.
The optimal treatment of insulin-dependent diabetes mellitus (IDDM), is the regulated delivery of insulin by functional beta cells. Pancreas transplantation, however, is a major surgical procedure with a high rate of complications. Transplantation of the isolated insulin-secreting islets of Langerhans is an alternative approach, which is easier and safer than whole organ transplantation. Clinical trials of islet transplantation have begun in a few specialized centers worldwide. Insulin independence at 1 year was achieved in 8% of the patients, but 20% of cases showed a graft function with a normal basal C peptide and improved glycemic regulation.
Beta-cell transplantation has so far been restricted by the scarcity of human islet donors. This shortage could be alleviated by methods for the isolation and/or culture of beta-cell progenitors. Such cells might also be protected from immunological rejection and recurring autoimmunity by genetic manipulation. The combination of these approaches with immunoisolation devices holds the promise of a widely available cell therapy for treatment of IDDM in the near future.
The pancreas is composed of at least three types of differentiated tissue: the hormone-producing cells in islets (4 different cell types), the exocrine zymogen-containing acini, and the centroacinar cells, ductules and ducts (ductal tree). All of these cells appear to have a common origin during embryogenesis in the form of duct-like protodifferentiated cells. Later in life, the acinar and ductal cells retain a significant proliferative capacity that can ensure cell renewal and growth, whereas the islet cells become mitotically inactive.
During embryonic development, and probably later in life, pancreatic islets of Langerhans originate from differentiating epithelial stem cells. These stem cells are situated in the pancreatic ducts but are otherwise poorly characterized. Pancreatic islets contain four islet cell types: alpha, beta, delta and pancreatic polypeptide cells that synthesize glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. The early progenitor cells to the pancreatic islets are multipotential and coactivate all the islet-specific genes from the time they first appear. As development proceeds, expression of islet-specific hormones becomes restricted to the pattern of expression characteristic of mature islet cells.
The characterization of pre-islet cells is of great interest for the development of therapeutics to treat diseases of the pancreas, particularly IDDM. Model systems have been described that permit the study of these cells. For example, Gu and Sarvetnick (1993) Development 118:33-46 identify a model system for the study of pancreatic islet development and regeneration. Transgenic mice carrying the mouse xcex3-interferon gene linked to the human insulin promoter exhibit inflammatory-induced islet loss. Significant duct cell proliferation occurs in these mice, leading to a striking expansion of pancreatic ducts. Endocrine progenitor cells are localized in these ducts. This model provides a source of progenitor cells for further study.
The human epidermal growth factor receptor (HER or ErbB) family consists of four distinct members, including the epidermal growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors plays an important role in the regulation of cell proliferation, differentiation, and survival in several different tissues. Binding of a specific ligand to one of the ErbB receptors triggers the formation of specific receptor homo- and heterodimers, with ErbB2 being the preferred signaling partner. The ErbB receptor ligands represent a complex variety of molecules. The EGF receptor binds six known ligands, including EGF, TGFxcex1, heparin-binding EGF-like growth factor, amphiregulin, epiregulin and betacellulin. Other members of the ErbB receptor family appear to function primarily through interaction with the neuregulins, a family of EGF-like growth factors encoded by at least three different genes: NRG1 (NDF, heregulin, GGF, ARIA or SMDF), NRG2, NRG3 and NRG4. Alternative transcript splicing from the NRG1 and NRG2 genes results in the production of multiple neuregulin isoforms. Distinct isoforms can elicit distinct biological activities depending on the cellular context, thereby modulating growth and development independently.
The differential expression of genes by progenitor cells, as compared to their differentiated progeny, is of interest for the characterization and isolation of the progenitor cells. Where the differentially expressed genes encode a receptor for biologically active molecules, the marker may further provide information about factors that affect the growth or differentiation of the progenitor cells. Where such genes encode proteins such as transcription factors, the marker may provide information about regulated gene expression in the progenitor cells.
Relevant Literature
The expression of ErbB2 in rat embryonic pancreas has been reported by LeBras et al. (1998) Diabetologia 41:1474-1481. Press et al. (1990) Oncogene 5:953-962 found that ErbB2 was not significantly expressed in the adult pancreas, though weak staining was seen in the ducts. Sundaresan et al. (1998) Endocrinology 139:4756-4764 have also reported expression of ErbB receptor and ligand in a ductal epithelial cell derived from rat embryos.
Hall et al. (1990) J Pathol 161(3):195-200; and Dugan et al. (1997) Pancreas 14(3):229-36 investigate the expression of ErbB2 in pancreatic cancers. The receptor has been found to be amplified and overexpressed in a number of human adenocarcinomas. The data suggest that there is abnormal expression of c-erb B-2 oncogene in about 20 per cent of cases, although mutational activation was not seen in human pancreatic adenocarcinoma.
Oberg-Welsh and Welsh (1996) Pancreas 12:334-339 studied the expression of protein tyrosine kinases in different preparations of insulin producing cells by polymerase chain reaction (PCR). Among the tyrosine kinases thus identified were the fibroblast growth factor receptor-4 (FGFR-4), c-kit, the insulin-like growth factor (IGF-I) receptor, and the cytoplasmic tyrosine kinase Jak2, which associates with the activated receptor for growth hormone (GH). Fetal islet-like structures were cultured in the absence or presence of the ligands to these receptors.
Transcription factors important for insulin gene expression are critical for development of the pancreas during embryogenesis (see Sander and German (1997) J. Mol. Med. 75:327-340). PDX-1 is one important marker. Like PDX-1, MSX-2 is a homeobox-containing transcription factor. It is part of a conserved family of transcription factors that play critical roles in tissue patterning and organogenesis during development. Msx-2 is expressed at a wide variety of sites in the developing embryo, but no specific role is known for pancreatic development (see Davidson (1995) Trends Genet. 11:405-411). It is not expressed in the normal adult pancreas (Maas et al. (1996) Ann. NY Acad. Sci. 785:171-181).
Polypeptide markers are provided that are expressed by progenitors of pancreatic islet cells. During regeneration of pancreatic islets, receptors of the ErbB family are expressed, including ErbB2, ErbB3 and ErbB4. Transcriptional factors are also shown to be expressed, including the homeobox-containing factor Msx-2. These markers are useful in identifying progenitor cells in the lineage that produces pancreatic islet cells; and can also be used in the detection of pancreatic islet regeneration. The progeny islet cells include insulin producing beta cells, and glucagon producing alpha cells.
Those markers that are expressed on the cell surface are useful for the enrichment of islet progenitor cells from complex cell mixtures. Such progenitor populations are useful in transplantation, for experimental evaluation, and as a source of lineage and cell specific products, including mRNA species useful in identifying genes specifically expressed in these cells, and as targets for the discovery of factors or molecules that can affect them. Cultures of such cells may utilize ligands that specifically interact with the cell surface markers. Ligands of interest include the neuregulins: NRG1, NRG2 and NRG3.